Cell ablation using trans-splicing ribozymes

ABSTRACT

The design of new ribozymes capable of self-catalyzed trans-splicing which are based upon the catalytic core of a Group I intron are described. Using this design, it is possible to construct ribozymes capable of efficiently splicing a new 3&#39; exon sequence into any chosen target RNA sequence in a highly precise manner. A method of cell ablation is also described that provides a toxic product to a host cell in vivo in a targetted, regulated manner utilizing novel trans-splicing ribozymes of the invention. Inactive pro-ribozyme forms are also described.

This application is a division of application Ser. No. 08/090,193, filedDec. 23, 1993, now U.S. Pat. No. 5,641,673, which is the U.S. NationalPhase of PCT/US92/00277 (Internations filing date Jan. 16, 1992), whichis a continuation-in-part of U.S. application Ser. No. 07/642,330, filedJan. 17, 1991 (abandoned).

FIELD OF THE INVENTION

The present invention is directed to novel trans-splicing ribozymes andmethods of cell ablation using these ribozymes.

BRIEF DESCRIPTION OF THE BACKGROUND ART

I. Group I Introns

RNA molecules with catalytic activity are called ribozymes or RNAenzymes (Cech, T. R., Ann. Rev. Biochem. 59:543-568 (1990). TheTetrahymena thermophila precursor rRNA contains an intron (a ribozyme)capable of catalyzing its own excision. This ribozyme is one of a classof structurally related Group I introns.

The splicing activity of the modified T. thermophila intron requires thepresence of a guanosine cofactor and a divalent cation, either Mg⁺⁺ orMn⁺⁺, and occurs via two sequential transesterification reactions (FIG.1). First, a free guanosine is bound to the ribozyme and its 3' hydroxylgroup is positioned to attack the phosphorus atom at the 5' splice site.The guanosine is covalently attached to the intron sequence and the 5'exon is released. Second, the phosphodiester bond located at the 3'splice site undergoes attack from the newly freed 3' hydroxyl group ofthe 5' exon, resulting in production of the ligated exon sequences. Theexcised intron subsequently undergoes a series of transesterificationreactions, involving its 3' hydroxyl group and internal sequences,resulting in the formation of shortened circular forms.

These successive reactions are chemically similar and appear to occur ata single active site. The reactions of self-splicing are characterizedby the formation of alternative RNA structures as differing RNA chainsare each brought to form similar conformations around the highlyconserved intron. Splicing requires the alignment of the intron-exonjunctions across a complementary sequence termed the "internal guidesequence" or IGS.

The first cleavage at the 5' splice site requires the formation of abase-paired helix (P1) between the IGS and sequences adjacent the splicesite. The presence of a U:G "wobble" base-pair within this helix definesthe phosphodiester bond that will be broken in the catalytic reaction ofthe ribozyme. After cleavage of this bond, a portion of the P1 helix isdisplaced and a new helix, P10, is formed due to complementarity betweenthe IGS and sequences adjacent the 3' splice site. An invariantguanosine residue precedes the phosphodiester at the 3' splice site,similar to the portion of the P1 sequence that it is displacing. Thus,ligation of the exons occurs in a reverse of the first cleavage reactionbut where new exon sequences have been substituted for those of theintron. It may be noted that intron circularization reactions subsequentto exon ligation also involve base-pairing of 5' sequences across theIGS, and attack mediated by the 3' hydroxyl group of the intron'sterminal guanine residue (Been, M.D. et al., "Selection OfCircularizaton Sites In A Group I IVS RNA Requires Multiple AlignmentsOf An Internal Template-Like Sequence," Cell 50:951 (1987)).

II. Catalytic Activities

In order to better define the structural and catalytic properties of theGroup I introns, exon sequences have been stripped from the "core" ofthe T. thermophila intron. Cech, T. R. et al., WO 88/04300, describes atleast three catalytic activities possessed by the Tetrahymena intronribozyme: (1) a dephosphorylating activity, capable of removing the 3'terminal phosphate of RNA in a sequence-specific manner, (2) an RNApolymerase activity (nucleotidyl transferase), capable of catalyzing theconversion of oligoribonucleotides to polyribonucleotides, and (3) asequence-specific endoribonuclease activity.

Isolated ribozyme activities can interact with substrate RNAs in trans,and these interactions characterized. For example, when truncated formsof the intron are incubated with sequences corresponding to the 5'splice junction, the site undergoes guanosien-dependent cleavage inmimicry of the first step in splicing. The substrate andendoribonucleolytic intron RNAs base-pair to form helix P1, and cleavageoccurs after a U:G base-pair at the 4th-6th position. Phylogeneticcomparisons and mutational analyses indicate that the nature of thesequences immediately adjacent the conserved uracil residue at the 5'splice site are unimportant for catalysis, provided the base-pairing ofhelix P1 is maintained (Doudna, J. A. et al., Proc. Natl. Acad. Sci. USA86: 7402-7406 (1989)).

The sequence requirements for 3' splice-site selection appear to liemainly within the structure of the intron itself, including helix P9.0and the following guanosine residue which delineates the 3' intronboundary. However, flanking sequences within the 3' exon are requiredfor the formation of helix P10 and efficient splicing, as shown bymutational analysis (Suh, E. R. et al., Mol. Cell. Biol. 10:2960-2965(1990)). In addition, oligonucleotides have been ligated in trans, usinga truncated form of the intron, and "external" guide sequence andoligonucleotides which had been extended by a 5' guanosine residue. Thesubstrate oligonucleotides corresponding to 3' exon sequences werealigned solely by the formation of P10-like helices on an externaltemplate, prior to ligation (Doudna, J. A. et al., Nature 339:519-522(1989)).

The cleavage activity of ribozymes has been targeted to specific RNAs byengineering a discrete "hybridization" region into the ribozyme, suchhybridization region being capable of specifically hybridizing with thedesired RNA. For example, Gerlach, W. L. et al., EP 321,201, constructeda ribozyme containing a sequence complementary to a target RNA.Increasing the length of this complementary sequence increased theaffinity of this sequence for the target. However, the hybridizing andcleavage regions of this ribozyme were integral parts of each other.Upon hybridizing to the target RNA through the complementary regions,the catalytic region of the ribozyme cleaved the target. It wassuggested that the ribozyme would be useful for the inactivation orcleavage of target RNA in vivo, such as for the treatment of humandiseases characterized by the production of a foreign host's RNA.However, ribozyme-directed trans-splicing, (as opposed totrans-cleavage) was not described or suggested.

The endoribonuclease activities (the cleavage activities) of variousnaturally-occurring ribozymes have been extensively studied. Analysis ofthe structure and sequence of these ribozymes has indicated that certainnucleotides around the cleavage site are highly conserved but flankingsequences are not so conserved. This information has lead to the designof novel endoribonuclease activities not found in nature. For example,Cech and others have constructed novel ribozymes with altered substratesequence specificity (Cech, T. R. et al., WO 88/04300; Koizumi, M. etal., FEBS Lett. 228:228-230 (1988); Koizumi, M. et al., FEBS Lett.239:285-288 (1988); Haseloff, J. et al., Nature 334:585-591 (1987); andHeus, H. A. et al., Nucl. Acids Res. 18:1103-1108 (1990)). From earlystudies of the self-cleaving plant viroids and satellite RNAs (Buzayan,J. M. et al., Proc. Natl. Acad. Sci. USA 83:8859-8862 (1986), guidelinesfor the design of ribozymes that are capable of cleaving other RNAmolecules in trans in a highly sequence specific have been developed(Haseloff, J. et al., Nature 334:585-591 (1988)). However, theseconstructs were unable to catalyze efficient, targeted trans-splicingreactions.

The joining of exons contained on separate RNAs, that is,trans-splicing, occurs in nature for both snRNP-mediated andself-catalyzed group I and group II introns. In trypanosome andCaenorhabditis elegans mRNAs, common 5' leader sequences are transcribedfrom separate genes and spliced to the 3' portions of the mRNAs(Agabian, N., Cell 61:1157-1160 (1990); Hirsh, D. et al., Mol. Biol.Rep. 14:115 (1990). These small "spliced leader" RNAs (slRNAs) consistof the 5' exon fused to sequences that can functionally substitute forU1 snRNA in mammalian snRNP-splicing extracts.

Also, both the group I and group II self-splicing introns are capable ofexon ligation in trans in artificial systems (Been, M. D. et al., Cell47:207-216 (1986); Galloway-Salvo, J. L. et al., J. Mol. Biol.211:537-549 (1990); Jacquier, A. et al., Science 234:1099-1194 (1986);and Jarrell, K. A. et al., Mol. Cell Biol. 8:2361-2366 (1988)).Trans-splicing occurs in vivo for group II introns in split genes ofchloroplasts (Kohchi, T. et al., Nucl. Acids Res. 16:10025-10036(1988)), and has been shown for a group I intron in an artificiallysplit gene in Escherichia coli (Galloway-Salvo, J. L. et al., J. Mol.Biol. 211:537-549 (1990)). In the latter case, a bacteriophage T4thymidylate synthase gene (td) containing a group I intron was dividedat the loop connecting the intron helix P6a. Transcripts of the td genesegments were shown to undergo trans-splicing in vitro, and to rescuedysfunctional E. coli host cells. Known base-pairings (P3, P6 and P6a)and possible tertiary interactions between the intron segments, allowedcorrect assembly and processing of the gene halves.

In vitro, the Tetrahymena ribozyme is capable of catalyzing thetrans-splicing of single-stranded model oligoribonucleotide substrates.Four components were necessary: ribozyme, 3' single-stranded RNA, 5'exon and GTP. A shortened form of the Tetrahymena ribozyme (L-21 ScaIIVS RNA), starting at the internal guide sequence and terminating atU₄₀₉ has been used in such a reaction (Flanegan, J. B. et al., J. Cell.Biochem. (Supp.)12 part D:28 (1988)). Attack by GTP at the 5' splicesite released the 5' exon which was then ligated by the ribozyme to the3' exon in a transesterification reaction at the 3' splice site.

The in vivo use of ribozymes as an alternative to the use of antisenseRNA for the targeting and destruction of specific RNAs has been proposed(Gerlach, W. L. et al., EP321,201; Cotten, M., Trends Biotechnol.8:174-178 (1990); Cotten, M. et al., EMBO J. 8:3861-3866 (1989); Sarver,N. et al., Science 47:1222-1225 (1990)). For example, expression of aribozyme with catalytic endonucleolytic activity towards an RNAexpressed during HIV-1 infection has been suggested as a potentialtherapy against human immunodeficiency virus type 1 (HIV-1) infection(Sarver, N. et al., Science 247:1222-1225 (1990); Cooper, M., CDC AIDSWeekly, Apr. 3, 1989, page 2; Rossi, J. J., Abstract of Grant No.1RO1AI29329 in Dialog's Federal Research in Progress File 265). However,such attempts have not yet been successful.

In a study designed to investigate the potential use of ribozymes astherapeutic agents in the treatment of human immunodeficiency virus type1 (HIV-1) infection, ribozymes of the hammerhead motif (Hutchins, C. J.et al., Nucl. Acids Res. 14:3627 (1986); Keese, P. et al., in Viroidsand Viroid-Like Pathogens, J. S. Semancik, ed., CRC Press, Boca Raton,Fla., 1987, pp. 1-47) were targeted to the HIV-1 gag transcripts.Expression of the gag-targeted ribozyme in human cell cultures resultedin a decrease (but not a complete disappearance of) the level of HIV-1gag RNA and in antigen p24 levels (Sarver, N. et al., Science 20247:1222-1225 (1990)). Thus, the medical effectiveness of Sarver'sribozyme was limited by its low efficiency since any of the pathogen'sRNA that escapes remains a problem for the host.

Another problem with in vivo ribozyme applications is that a highribozyme to substrate ratio is required for ribozyme inhibitory functionin nuclear extracts and it has been difficult to achieve such ratios.Cotton et al. achieved a high ribozyme to substrate ration bymicroinjection of an expression cassette containing a ribozyme-producinggene operably linked to a strong tRNA promoter (a polymerase IIIpromoter) in frog oocytes, together with substrate RNA that contains thecleavage sequence for the ribozyme (Cotton, M. et al., EMBO J.8:3861-3866 (1989). However, microinjection is not an appropriate methodof delivery in multicellular organisms.

The in vivo activity of ribozymes designed against mRNA coding forEscherichia coli β-galactosidase has been reported (Chuat, J.-C. et al.,Biochem. Biophys. Res. Commun. 162:1025-1029 (1989)). However, thisactivity was only observed when the ribozyme and target were transfectedinto bacterial cells on the same molecule. Ribozyme activity wasinefficient when targeted against an mRNA transcribed from a bacterial Frepisome that possessed the target part of the β-galactosidase gene.

Thus, current technological applications of ribozyme activities arelimited to those which propose to utilize a ribozyme's cleavage activityto destroy the activity of a target RNA. Unfortunately, suchapplications often require complete destruction of all target RNAmolecules, and/or relatively high ribozyme:substrate ratios to ensureeffectiveness and this has been difficult to achieve. Most importantly,the modified ribozymes of the art are not capable of efficient, directedtrans-splicing.

Accordingly, a need exists for the development of highly efficientribozymes and ribozyme expression systems. Especially, the art does notdescribe an effective means in which to destroy an existing RNA sequenceor to alter the coding sequence of an existing RNA by the trans-splicingof a new RNA sequence into a host's RNA.

SUMMARY OF THE INVENTION

Recognizing the potential for the design of novel ribozymes, andcognizant of the need for highly efficient methods to alter the geneticcharacteristics of higher eukaryotes in vivo, the inventors haveinvestigated the use of ribozymes to alter the genetic information ofnative RNA's in vivo. These efforts have culminated in the developmentof highly effective trans-splicing ribozymes, and guidelines for theengineering thereof.

According to the invention, there is first provided an RNA or DNAmolecule, such molecule encoding a trans-splicing ribozyme, suchribozyme being capable of efficiently splicing a new 3' exon sequenceinto any chosen target RNA sequence in a highly precise manner, in vitroor in vivo, and such molecule being novel in the ability to accomodate,any chosen target RNA or 3' exon sequences, and in the addition of acomplementary sequence which enhances the specificity of such ribozyme.

According to the invention, there is also provided an RNA or DNAmolecule, such molecule encoding a ribozyme, the sequence for suchribozyme being a fusion RNA, such fusion RNA providing a first RNAsequence that is sufficient for targeting such ribozyme to hybridize toa target RNA, and further a second RNA sequence, such second RNAsequence capable of being transposed into the target RNA, and suchsecond RNA sequence encoding an RNA sequence foreign to the targeted RNAsequence.

According to the invention, there is further provided an RNA or DNAmolecule, such molecule encoding a ribozyme, the sequence for suchribozyme being a fusion RNA as described above, the first RNA sequenceprovided by the fusion RNA being a sequence for targeting such RNAmolecule to hybridize to GAL4 RNA, and the second RNA sequence of thefusion RNA providing the coding sequence of the A chain of diphtheriatoxin (DTA).

According to the invention, there is also provided an RNA or DNAmolecule, such molecule encoding a conformationally disrupted ribozymeof the invention, a pro-ribozyme, such pro-ribozyme beingsubstrate-activated that is, such pro-ribozyme possessing neglible or noself-cleavage or trans-splicing activity, until being reactived byspecific interaction with target RNA.

According to the invention, there is further provided an RNA or DNAmolecule containing a ribozyme or pro-ribozyme expression cassette, suchcassette being capable of being stably maintained in a host, or insertedinto the genome of a host, and such cassette providing the sequence of apromoter capable of functioning in such host, operably linked to thesequence of a ribozyme or pro-ribozyme of the invention.

According to the invention, there is further provided an RNA or DNAmolecule containing a ribozyme or pro-ribozyme expression cassette, suchcassette being capable of being stably inserted into the genome of ahost, such ribozyme expression cassette providing the sequence of aGAL4-responsive promoter operably linked to the sequence of a ribozymeor pro-ribozyme of the invention.

According to the invention, there is further provided a method forin-vitro trans-splicing, such method comprising the steps of (1)providing a ribozyme or pro-ribozyme of the invention and an appropriatesubstrate for such ribozyme in vitro, (2) further providing in vitroreaction conditions that promote the desired catalytic activity of suchribozyme or pro-ribozyme; and (3) allowing such ribozyme or pro-ribozymeto react with such substrate under such conditions.

According to the invention, there is further provided a method for invivo trans-splicing, such method comprising the steps of (1) providingan RNA or DNA molecule of the invention to a host cell, (2) expressingthe ribozyme or pro-ribozyme encoded by such molecule in such host cell,(3) expressing a substrate of such ribozyme or pro-ribozyme in such hostcell, and (4) allowing such ribozyme or pro-ribozyme to react with suchsubstrate in such host cell.

According to the invention, there is further provided a method forinactivating the activity of a target RNA, such method comprising (1)providing a ribozyme or pro-ribozyme of the invention, such ribozyme orpro-ribozyme being catalytically active against such target RNA, (2)providing such target RNA, and (3) providing conditions that allow suchribozyme or pro-ribozyme to express its catalytic activity towards suchtarget RNA.

According to the invention, there is further provided a method forproviding a desired genetic sequence to a host cell in vivo, such methodcomprising (1) providing a ribozyme or pro-ribozyme of the invention toa desired host cell, such ribozyme or pro-ribozyme being catalyticallyactive against a target RNA in such host cell, (2) providing suchribozyme or pro-ribozyme encoding such desired genetic sequence, and (3)providing conditions that allow such ribozyme or pro-ribozyme totrans-splice such desired genetic sequence into the sequence of thetarget RNA.

According to the invention, there is further provided a method for cellablation in multicellular plants and animals, such method comprisingproviding a ribozyme or pro-ribozyme of the invention to a any hostcell, and especially into a fertilized embryonic host cell, suchribozyme or pro-ribozyme encoding the sequence of a gene toxic to suchhost cell and such ribozyme or pro-ribozyme being capable oftrans-splicing with a desired target in such host cell.

According to the invention, there is further provided a method forengineering male or female sterility in agronomically important plantspecies, such method comprising the ablation of any cell necessary forfertility using a ribozyme or pro-ribozyme of the invention.

According to the invention, there is further provided a method ofimmunizing plants against plant pathogens, such method comprising theconstruction of transgenic plants capable of expressing a plantpathogen-specific fusion ribozyme or pro-ribozyme of the invention, andsuch ribozyme or pro-ribozyme being capable of ablating any host cellinfected with such pathogen.

According to the invention, there is further provided a transformed,pathogen-resistant microorganism, such microorganism being resistant toa desired pathogen, such microorganism being transformed with a ribozymeor pro-ribozyme of the invention and such ribozyme or pro-ribozymeproviding a catalytic activity that targets a nucleic acid moleculeexpressed by such pathogen.

According to the invention, there is further provided a viral pathogencapable of delivering a desired ribozyme or pro-ribozyme activity to adesired host, such ribozyme or pro-ribozyme activity being delivered bya ribozyme or pro-ribozyme of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the mechanism of ribozyme splicing of the group Iintron.

FIG. 2 is a diagram of structure of the design of ribozymes fortrans-splicing. FIG 2A depicts Tetrahymena thermophila self-splicingrRNA intron; FIG. 2B depicts Target mRNA and trans-splicing ribozyme orpro-ribozyme of the invention.

FIG. 3A is a diagram of the design of a CAT-LacZ α-peptidetrans-splicing ribozyme; top structure depicts Tetrahymena thermophilaself-splicing rNRA intron; bottom structure depicts CAT-LacZtrans-splicing ribozyme; FIG 3B is the complete DNA coding sequence ofthe CAT-LacZ ribozyme.

FIGS. 4A-4C present the sequences of cucumber mosaic virus (CMV) RNA 4trans-splicing ribozymes. FIG. 4A depicts virus RNA target sequences;FIG 4B depicts Oligonucleotide target sequences; FIG. 4C depicts CMVRNA4--diphtheria toxin A-chain trans-splicing ribozymes.

FIG. 5 is a comparison of cucumber mosaic virus 3/4 sequences.

FIG. 6A is a diagram of the design of a Gal4-Diphtheria toxin A (DTA)chain trans-splicing ribozyme; FIG 6B: top structure depicts Tetrahymenathermophila self-splicing rRNA intron; bottom structure depictsGal4-Dt-A trans-splicing ribozyme; is the complete coding sequence ofthe Gal4-DTA ribozyme with the isoleucine substitution.

FIG. 7 is a diagram of the P-element mediated "enhancer-trapping" methodfor expression of Gal4 protein.

FIG. 8 presents a partial sequence of wild-type DTA and DTA 3' exonmutants and depicts prevention of toxin expression from splicingby-products.

FIG. 9 is a map of Gal4 vector (pGaTB and pGaTN). Unique sites areindicated in italics; 3' NotI site is unique in pGatB only.

FIG. 10 is a map of Gal UAS vector (pUAST). Unique sites are indicatedin italics.

FIG. 11 is a cuticle preparation of a Drosophila embryos expressing aGal4-DTA trans-splicing ribozyme.

FIGS. 12A-12D present the rationale for "pro-ribozyme" design. Arrowsshow sites of ribozyme cleavage, "antisense" regions are shown in black,catalytic domains are shown with radial shading, and 3' "exon" sequencesare shown with light shading. In the absence of the target mRNA,trans-splicing ribozymes may transiently base-pair, and react withheterologous sequences (including their own). In addition, scission atthe "3' exon" junction will occur. Inactive "pro-30 ribozymes" areconstructed to contain extra self-complementary sequences which causethe catalytic center of the ribozyme to be mis-folded. Active ribozymesare only formed after base-pairing with the intended target mRNA--andconsequent displacement of the interfering secondary structure.

FIGS. 13, and 13A-13C show the sequence and predicted secondarystructure of the CAT-LacZ trans-splicing ribozyme. In FIG. 13C ribozyme"core" sequences are shaded (after Cech, Gene 73:259-271 (1988)).Helices P8 are shown for the unmodified ribozyme and pro-ribozymes 1 and2, with 13 and 18 nucleotides, respectively, of sequence complementaryto the "antisense", region (highlighted).

FIG. 14A shows active CAT-LacZ α-peptide trans-splicing ribozyme shownschematically, with "antisense", ribozyme domain with helix P8 and 3'"exon" sequences; (2) (a) inactive CAT-LacZ α-peptide transs-splicingpro-ribozyme shown with base-pairing between sequences in the modifiedhelix P8 and the "anti-sense" region; and FIG. 14B, bottom, shows theactive CAT-LacZ α-peptide trans-splicing pro-ribozyme, afterbase-pairing with the CAT mRNA, displacement of the helixP8--"antisense" pairing, and re-formation of helix P8.

FIG. 15 shows stability of CAT-LacZ pro-ribozyme transcripts. Plasmidscontaining the CAT-LacZ ribozyme and pro-ribozyme sequences were cleavedwith EcoRI and transcribed using T7 or SP6 RNA polymerase and 32-P!UTP.Radiolabeled transcripts were fractionated by 5% polyacrylamide gelelectrophoresis in 7M urea and 25% formamide, and autoradiographed. Theribozyme transcripts underwent extensive hydrolysis, primarily at the"3' exon" junction. The pro-ribozyme forms were markedly less reactive.

FIG. 16 shows endoribonuclease activity of CAT-LacZ pro-ribozymes.Plasmids containing CAT-LacZ ribozyme and pro-ribozyme sequences werecleaved, with ScaI, and transcribed with T7 or SP6 RNA polymerase.

Transcripts were incubated for 30' at 37° C., 45° C. and 50° C. in 40 mMTris-HCl pH 7.5, 6 mM MgCl₂, 2 mM spermidine, 10 mM NaCl, 2 mM GTP withradiolabeled CAT RNA, transcribed using T7 RNA polymerase from plasmidcut with PuvII. Products were fractionated by 5% polyacrylamide gelelectrophoresis in 7M urea and 25% formamide, and autoradiographed. RNAmediated cleavage of the 173 nt (nucleotides) CAT RNA produces 5' and 3'fragments of 76 nt and 97 nt, respectively.

FIG. 17 shows the "wild-type" and modified helices P8 used forpro-ribozyme design with possible base-pairs indicated in schematicform. Those bases which are complementary to the "anti-sense" portion ofthe corresponding pro-ribozyme, are shown in bold type. The number ofcomplementary bases is listed next to each helix. The helices areordered by the stability of the corresponding pro-ribozyme transcripts,as measured by the degree of "3' exon" hydrolysis during in vitrotranscription.

FIG. 18 shows the stability of GAL4-DTA pro-ribozymes. Plasmidscontaining ribozyme and pro-ribozyme sequences were linearized with XhoIand transcribed using T7 RNA polymerase. Transcripts were incubated for60' at 50° C. n 40 mM Tris-HCl pH 7.5, 6 mM mgCl₂, 2 mM spermidine, 10nM NaCl, 1 mM GTP, were fractionated by 5% polyacrylamide gelelectrophoresis in 7M urea and 25% formamide, and autoradiographed.Ribozyme transcripts are extensively hydrolysed under these conditions,while pro-ribozyme 1 is less so and pro-ribozyme 2 is stable.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

In the description that follows, a number of terms used in recombinantDNA (rDNA) technology are extensively utilized. In order to provide aclear and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

Ribozyme. An RNA molecule that inherently possesses catalytic activity.

Trans-splice. A form of genetic manipulation whereby a nucleic acidsequence of a first polynucleotide is co-linearly linked to or insertedinto the sequence of a second polynucleotide, in a manner that retainsthe 3'→5' phosphodiester linkage between such polynucleotides. By"directed" trans-splicing or "substrate-specific" trans-splicing ismeant a trans-splicing reaction that requires a specific specie of RNAas a substrate for the trans-splicing reaction (that is, a specificspecie of RNA in which to splice the transposed sequence). Directedtrans-splicing may target more than one RNA specie if the ribozyme orpro-ribozyme is designed to be directed against a target sequencepresent in a related set of RNAs.

Target RNA. An RNA molecule that is a substrate for the catalyticactivity of a ribozyme or pro-ribozyme of the invention.

Expression Cassette. A genetic sequence that provides sequencesnecessary for the expression of a ribozyme or pro-ribozyme of theinvention.

Stably. By "stably" inserting a sequence into a genome is intendedinsertion in a manner that results in inheritance of such sequence incopies of such genome.

Operable linkage. An "operable linkage" is a linkage in which a sequenceis connected to another sequence (or sequences) in such a way as to becapable of altering the functioning of the sequence (or sequences). Forexample, by operably linking a ribozyme or pro-ribozyme encodingsequence to a promoter, expression of the ribozyme or pro-ribozymeencoding sequence is placed under the influence or control of thatpromoter. Two nucleic acid sequences (such as a ribozyme or pro-ribozymeencoding sequence and a promoter region sequence at the 5' end of theencoding sequence) are said to be operably linked if induction ofpromoter function results in the transcription of the ribozyme orpro-ribozyme encoding sequence and if the nature of the linkage betweenthe two sequences does not (1) result in the introduction of aframe-shift mutation, (2) interfere with the ability of the expressionregulatory sequences to direct the expression of the ribozyme. Thus, apromoter region would be operably linked to a nucleic acid sequence ifthe promoter were capable of effecting the synthesis of that nucleicacid sequence.

II. Engineering of the Ribozyme of the Invention

The trans-splicing ribozymes, pro-ribozymes and methods of the inventionprovide, for the first time, a ribozyme capable of directedtrans-splicing into any RNA sequence, and especially into mature(non-intron-containing) mRNA. The trans-splicing ribozyme as describedherein, with its extended complementarity to the target, greatly differsfrom T. thermophila derived endoribonuclease activities described in theart. The additional complementarity of the ribozymes of the inventionconfers increased affinity and specificity for the target and thecomplementarity is not an integral part of the catalytic activity. Inaddition, cleavage occurs efficiently and precisely in the absence ofdenaturants and at high concentrations of Mg⁺⁺.

The guidelines described herein for the design of trans-splicingribozymes are conservative, based on the well characterized propertiesof group I self-splicing introns and are meant to provide a generalscheme for the design of any directed trans-splicing ribozyme.Accordingly, the guidelines presented herein are not limited to thegroup I intron of the T. thermophila pre-mRNA and may be used by one ofskill in the art to design a ribozyme of the invention with other groupI introns using such guidelines and knowledge in the art.

The native T. thermophila ribozyme (the intron sequence) is located frombase 53 to base 465 in the sequence below of the T. thermophilaextrachromosomal rDNA:

    __________________________________________________________________________    TGACGCAATT            CAACCAAGCG                    CGGGTAAACG                            GCGGGAGTAA                                    CTATGACTCT    CTAAATAGCA            ATATTTACCT                    TTGGAGGGAA                            AAGTTATCAG                                    GCATGCACCT    CCTAGCTAGT            CTTTAAACCA                    ATAGATTGCA                            TCGGTTTAAA                                    AGGCAAGACC    GTCAAATTGC            GGGAAAGGGG                    TCAACAGCCG                            TTCAGTACCA                                    AGTCTCAGGG    GAAACTTTGA            CATGGCCTTG                    CAAAGGGTAT                            GGTAATAAGC                                    TGACGGACAT    GGTCCTAACC            ACGCAGCCAA                    GTCCTAAGTC                            AACAGATCTT                                    CTGTTGATAT    GGATGCAGTT            CACAGACTAA                    ATGTCGGTCG                            GGGAAGATGT                                    ATTCTTCTCA    TAAGATATAG            TCGGACCTCT                    CCTTAATGGG                            AGGTAGCGGA                                    TGAATGGATG    CAACACTGGA            GCCGCTGGGA                    ACTAATTTGT                            ATGCGAAAGT                                    ATATTGATTA    GTTTTGGAGT            ACTCGTAAGG                    TAGCCAAATG                            CCTCGTCATC                                    TAATTAGTGA    CGCGCATGAA            TGGATTA  SEQ ID NO.1!    __________________________________________________________________________

(Kan, N. C. et al., Nucl. Acids Res. 30:2809-2822 (1982)).

As described herein, the directed trans-splicing ribozymes of theinvention are engineered using the catalytic core of this intron. Theintron, and its catalytic core can be isolated by methods known in theart. The catalytic core of the intron, that is, the truncated intron,differs form the full-length intron only in that it is truncated at theScaI site, thus removing the last five nucleotides of the intron. Thetruncated intron RNA may be prepared by techniques known in the art ormay be purchased commercially in kit form from commercial sources suchas, for example, product #72000 from US Biochemical, Cleveland, Ohio(RNAzyme™ Tet 1.0 Kit). This US Biochemical kit provides ribozyme andthe protocol for the use of the ribozyme. Transcribed Tet.1 cDNA may beused as the substrate for polymerase chain reaction (PCR) mutagenesis asdescribed below, to produce a synthetic trans-splicing enzyme.

Substrate specificity of the ribozyme of the invention, that is, theability of the ribozyme to "target" a specific RNA as a substrate, isconferred by fusing complementary sequences specific to the target(substrate) RNA to the 5' terminus of the ribozyme.

Directed trans-splicing specificity of the ribozyme of the invention,that is, specificity in trans-splicing a desired foreign sequence ofinterest with the sequence of a target RNA, is conferred by providing anew 3' exon at the 3' terminus of the ribozyme. Details of the designare further provided below.

To alter the structural and catalytic properties of the Group I introns,exon sequences replace the flanking sequence of such introns so thatonly the catalytic core of the intron, the ribozyme, remains. Theresulting modified ribozyme can interact with substrate RNAs in trans.When truncated forms of the intron (i.e., the catalytic "core," i.e.truncated at the ScaI site, removing the last five nucleotides of theintron) are incubated with sequences corresponding to the 5' splicejunction of the native ribozyme, the site undergoes guanosine-dependentcleavage in mimicry of the first step in splicing.

Engineering of the ribozymes of the invention requires consideration ofthe four guidelines that follow.

First, a splice site must be chosen within the target RNA. In the finaltrans-splicing complex, only the 5' portion of the P1 duplex iscontributed by the target RNA. Only a single conserved residue, uracil,is required immediately 5' of the intended splice site. This is the solesequence requirement in the target RNA. There is no inate structurerequired of the target RNA. Mature NRNA may be targeted and thetrans-splicing reaction performed in the cell's cytoplasm rather than inthe nucleus against pre-mRNA. This obviates the need for highconcentrations of ribozyme in a cell's nucleus.

Second, having chosen a particular target sequence, compensatingsequence changes must be added to the 5' section of the ribozyme inorder to allow the formation of a suitable helix P1 between the targetand ribozyme RNAs. It is highly desired is that the helix P1 shouldcontain a U:G base-pair at the intended 5' splice site, and should bepositioned at the 4th, 5th (preferred) or 6th position from the base ofthe helix (Doudna, J. A., et al., "RNA Structure, Not SequenceDetermines The 5' Splice-Site Specificity of a Group I Intron," Proc.Natl. Acad. Sci. USA 86:7402-7406 (1989), incorporated herein byreference). For the native T. thermophila intron, P1 extends for anadditional 3 base pairs past the intended 5' splice site, and, in apreferred embodiment, this is maintained in the trans-splicing ribozymeof the invention. For trans-splicing to be efficient, the substrate andendoribonucleolytic intron RNAs must base-pair to form helix P1, with aresulting wobble U:G base-pair. Cleavage of the target RNA occurs at thephosphodiester bond immediately 3' to (after the) U:G base-pair.Phylogenetic comparisons and mutational analyses indicate that thenature of the sequences immediately adjacent the conserved uracilresidue at the 5' splice site are unimportant for catalysis, providedthe base-pairing of helix P1 is maintained.

Third, the exon sequences flanking the 3' splice site must be chosen,and adjustments made in the 5' section of the ribozyme, if necessary, toallow the formation of a stable P10 helix. While the P10 helix may bedispensesd with if necessary, its presence enhances splicing andpreferred embodiments of the ribozyme of the invention retain the P10helix (Suh, E. R. et al., "Base Pairing Between The 3' Exon And AnInternal Guide Sequence Increases 3' Splice Site Specificity in theTetrahymena Self-Splicing rRNA Intron," Mol. Cell. Biol. 10:2960-2965(1990)). The helices P1 and P10 overlap along the T. thermophila intronIGS, and the 2nd and 3rd residues following both the 5' and 3' splicesites are complementary to the same residues in the IGS (FIG. 2). Whilethere may be some advantage in following this, many natural group Iintrons do not share this constraint, so the choice of 3' exon sequencesmay be determined primarily by experimental considerations. Suchconsiderations reflect the wide flexibility in choice of splice sites.For example, if it is desired to join two sequences at a given point,the sequence at such point cannot be mutated or otherwise altered by thetrans-splicing event. Either P1 or P10 can be made shorter if theoverlapping sequences don't otherwise accomodate for the desired splicesite.

The sequence requirements for 3' splice-site selection appear to liemainly within the structure of the intron (the ribozyme) itself,including helix P9.0 and the adjoining 3' guanosine residue whichdelineates the 3' intron boundary. P9.0 is wholly contained within theintron sequences and helps define the adjacent 3' splice site. For thetrans-splicing design, the P9.0 helix and the rest of the functional RNAelements within the intron are not altered. The structuralcharacteristics of the P9.0 helix are known (Michel, F. et al., "TheGuanosine Binding Site of the Tetrahymena Ribozyme," Nature 342:391-395(1989)). However, flanking sequences within the 3' exon are required forthe formation of helix P10 and efficient splicing, as shown bymutational analysis.

Fourth, a region of complementary sequence is placed at the 5' terminusof the trans-splicing ribozyme in order to increase its affinity andspecificity for the target RNA. As shown herein, an arbitrary length ofaround 40 residues has been used. Other lengths may be used providedthey are not detrimental to the desired effect.

For example, starting with the T. thermophila self-splicing intron(diagrammed below): ##STR1## (The "1" and "12" in the above diagram (andin other ribozyme diagrams throughout the application) note the firstand second splice sites, respectively.)

(1) a "5'" site is chosen adjacent to a uracil residue within a chosentarget RNA. The sequences involved in complementarity do not immediatelyabut sequences involved in P1 helix formation but are separated, forexample, by five nucleotides also involved in P10 formation;

(2) sequences complementary to the chosen RNA are fused to the 5'portion of the self-splicing Group I intron. Base-pairing betweenribozyme and target RNA allow formation the of the helix P1;

(3) the chosen "3' exon" sequences are fused to the 3' portion of theribozyme, maintaining the conserved helix P10; and

(4) to increase affinity for the target RNA, if desired, a section ofextended sequence complementarity is fused to the 5' portion of theribozyme to allow the formation of 30-40 base-pairs.

The alignment of the resulting trans-splicing ribozyme with its targetRNA may be diagrammed as shown immediately below. The target RNAsequence represents the top line. The ribozyme sequence is aligned belowit, a continuous sequence wrapping around the lower two lines whereinthe hybridization of the nucleotides at the 5' and 3' ends and P1 andP10 of the ribozyme may be seen. ##STR2##

According to the invention, trans-splicing ribozymes can be designedthat will trans-splice essentially any RNA sequence onto any RNA target.It is not necessary that the target contain an intron sequence or thatthe ribozyme be an intron in the target sequence. For example, astrategy for such design may include (1) the identification of thedesired target RNA (2) cloning and/or sequencing of the desired targetRNA or portion thereof (3) selection of a desired coding sequence totrans-splice into the target RNA, (4) the construction of a ribozyme ofthe invention capable of hybridizing to such target using the guidelinesherein and (5) confirmation that the ribozyme of the invention willutilize the target as a substrate for the specific trans-splicingreaction that is desired and (6) the insertion of the ribozyme into thedesired host cell.

Choice of a target RNA will reflect the desired purpose of thetrans-splicing reaction. If the purpose of the reaction is to inactivatea specific RNA, then such RNA must be trans-spliced at a position thatdestroys all functional peptide domains encoded by such RNA and at aposition that does not result in continued expression of the undesiredgenetic sequences. If more than one allele of the gene encoding such RNAexists, the ribozyme should preferably be designed to inactivate thetarget RNA at a site common to all expressed forms. Alternatively, morethan one ribozyme may be provided to the cell, each designed toinactivate a specific allelic form of the target RNA.

When only inactivation of the target RNA is desired, and not theexpression of a new, desired RNA sequence, it is not necessary that theforeign RNA donated by the ribozyme provide a sequence capable of beingtranslated by the host cell, and a sequence containing translationalstop codons may be used as a truncated intron, for example, the intronribozyme truncated at the ScaI site.

If the purpose of the trans-splicing reaction is to provide a genetictrait to a host cell, then the choice of target RNA will reflect thedesired expression pattern of the genetic trait. If it is desired thatthe genetic trait be continuously expressed by the host, then the targetRNA should also to be continuously expressed. If it is desired that thegenetic trait be selectively expressed only under a desired growth,hormonal, or environmental condition, then the target RNA should also beselectively expressed under such conditions.

It is not necessary that expression of the ribozyme itself beselectively limited to a desired growth, hormonal, or environmentalcondition if the substrate for such ribozyme is not otherwise present inthe host as the ribozyme itself is not translated by the host. Thus,sequences encoded by the RNA donated by the ribozyme of the inventionare not translated in a host until the trans-splicing event occurs andsuch event may be controlled by the expression of the ribozyme substratein the host.

If desired, expression of the ribozyme may be engineered to occur inresponse to the same factors that induce expression of a regulatedtarget, or, expression of the ribozyme may be engineered to provide anadditional level of regulation so as to limit the occurrence of thetrans-splicing event to those conditions under which both the ribozymeand target are selectively induced in the cell, but by differentfactors, the combination of those factors being the undesired event.Such regulation would allow the host cell to express the ribozyme'starget under those conditions in which the ribozyme itself was notco-expressed.

The sequence of the ribozyme domain that hybridizes to the target RNA isdetermined by the sequence of the target RNA. The sequence of the targetRNA is determined after cloning sequences encoding such RNA or aftersequencing a peptide encoded by such target and deducing an RNA sequencethat would encode such a peptide. Cloning techniques known in the artmay be used for the cloning of a sequence encoding a target RNA.

The selection of a desired sequence to be trans-spliced into the targetRNA (herein termed the "trans-spliced sequence") will reflect thepurpose of the trans-splicing. If a trans-splicing event is desired thatdoes not result in the expression of a new genetic sequence, then thetrans-spliced sequence need not encode a translatable protein sequence.If a trans-splicing event is desired that does result in the expressionof a new genetic sequence, and especially a new peptide or proteinsequence, then the trans-spliced sequence may further providetranslational stop codons, and other information necessary for thecorrect translational processing of the RNA in the host cell. If aspecific protein product is desired as a result of the trans-splicingevent then it would be necessary to maintain the amino acid readingframe in the resulting fusion.

The identification and confirmation of the specificity of a ribozyme ofthe invention is made by testing a putative ribozyme's ability tocatalyze the desired trans-splicing reaction only in the presence of thedesired target sequence. The trans-splicing reaction should not occur ifthe only RNA sequences present are non-target sequences to which suchribozyme should not be responsive (or less responsive). Suchcharacterization may be performed with the assistance of a marker suchthat correct (or incorrect) ribozyme activity may be more easilymonitored. In most cases it is sufficient to test the ribozyme againstits intended target in vitro and then transform a host cell with it forstudy of its in vivo effects.

When it is desired to eliminate a host's RNA, such elimination should beas complete as possible. When it is desired to provide a new geneticsequence to a host cell, the trans-splicing reaction of the inventionneed not be complete. It is an advantage of the invention that,depending upon the biological activity of the peptide that is translatedfrom such genetic sequence, the trans-splicing event may in fact bequite inefficient, as long as sufficient trans-splicing occurs toprovide sufficient mRNA and thus encoded polypeptide to the host for thedesired purpose.

Transcription of the ribozyme of the invention in a host cell occursafter introduction of the ribozyme gene into the host cell. If thestable retention of the ribozyme by the host cell is not desired, suchribozyme may be chemically or enzymatically synthesized and provided tothe host cell by mechanical methods, such as microinjection,liposome-mediated transfection, electroporation, or calcium phosphateprecipitation. Alternatively, when stable retention of the gene encodingthe ribozyme is desired, such retention may be achieved by stablyinserting at least one DNA copy of the ribozyme into the host'schromosome, or by providing a DNA copy of the ribozyme on a plasmid thatis stably retained by the host cell.

Preferably the ribozyme of the invention is inserted into the host'schromosome as part of an expression cassette, such cassette providingtranscriptional regulatory elements that will control the transcriptionof the ribozyme in the host cell. Such elements may include, but notnecessarily be limited to, a promoter element, an enhancer or UASelement, and a transcriptional terminator signal. Polyadenylation is notnecessary as the ribozyme is not translated. However, suchpolyadenylation signals may be provided in connection with the sequenceencoding the element to be trans-spliced.

Expression of a ribozyme whose coding sequence has been stably insertedinto a host's chromosome is controlled by the promoter sequence that isoperably linked to the ribozyme coding sequences. The promoter thatdirects expression of the ribozyme may be any promoter functional in thehost cell, prokaryotic promoters being desired in prokaryotic cells andeukaryotic promoters in eukaryotic cells. A promoter is composed ofdiscrete modules that direct the transcriptional activation and/orrepression of the promoter in the host cell. Such modules may be mixedand matched in the ribozyme's promoter so as to provide for the properexpression of the ribozyme in the host. A eukaryotic promoter may be anypromoter functional in eukaryotic cells, and especially may be any of anRNA polymerase I, II or III specificity. If it is desired to express theribozyme in a wide variety of eukaryotic host cells, a promoterfunctional in most eukaryotic host cells should be selected, such as arRNA or a tRNA promoter, or the promoter for a widely expressed mRNAsuch as the promoter for an actin gene, or a glycolytic gene. If it isdesired to express the ribozyme only in a certain cell or tissue type, acell-specific (or tissue-specific) promoter elements functional only inthat cell or tissue type should be selected.

The trans-splicing reaction is chemically the same whether it isperformed in vitro or in vivo. However, in vivo, since cofactors areusually already present in the host cell, the presence of the target andthe ribozyme will suffice to result in trans-splicing.

The trans-splicing ribozymes and methods of the invention are usful inproducing a gene activity useful for the genetic modification, and/orcell death, of targeted cells. For example, the trans-splicing reactionof the invention is useful to introduce a protein with toxic propertiesinto a desired cell. The susceptibility of cells will be determined bythe choice of the target RNA and the regulatory controls that dictateexpression of the ribozyme. For example, a ribozyme that transposes anRNA sequence encoding a toxic protein may be engineered so thatexpression of the ribozyme will depend upon the characteristics of anoperably-linked promoter. In a highly preferred embodiment, diptheriatoxin peptide A is encoded by that part of the ribozyme that istransposed into a desired target in the host. Conditional expression ofthe ribozyme and diphtheria toxin peptide A chain results in the deathof the host cell. Other useful peptide toxins include ricin, exotonin A,and herpes thymidine kinase (Evans, G. A., Genes & Dev. 3:259-263(1989)). In addition, various lytic enzymes have the potential fordisrupting cellular metabolism. For example, a fungal ribonuclease maybe used to cause male sterility in plants (Mariani, C. et al., Nature347:737-741 (1990)). Particular tissues might be destroyed due tolimited expression of the target RNA. Further, if a viral RNA is used astarget, new forms of virus resistance, or therapies may be engineered.

A binary system for control of tissue-specific gene expression and/orfor ectopic ablation may be designed using the ribozymes of theinvention. For example, lines of Drosophila that express the yeasttranscription activator GAL4 in a tissue and spatial-specific patternusing P-element enhancer-trap vectors may be used. Any transcriptionalactivator may be used in place of GAL4 and the invention is not intendedto be limited to GAL4. A gene encoding a fusion ribozyme that is capableof trans-splicing the DTA sequence may be placed under the control ofthe GAL4-UAS promoter and inserted into Drosophila in a geneticallystable manner. Such ribozymes will not be expressed in Drosophila in theabsence of GAL4. Accordingly, crossing Drosophila hosts geneticallycarrying this ribozyme construct with Drosophila hosts that express GAL4in a tissue-specific manner result in progeny hat, when GAL4 expressionis induced, exhibit a pattern of cell death similar to the pattern ofGAL4 expression.

In addition, by targetting the ribozyme to trans-splice with the GAL4mRNA, the splicing activity of the ribozyme inactivates GAL4 expressionand ribozyme expression may be self-regulated.

Pro-ribozymes

A trans-splicing ribozyme, as described above, consists of three fusedsequence elements--a 5' "anti-sense" region which is complementary tothe target RNA, the catalytic region which is based on a self-splicingGroup I intron, and 3' "exon" sequences. The 5' region can base pairwith the chosen target RNA, to bring it into proximity with thecatalytic sequences of the Group I intron. The structure of the Group Iintron provides a chemical environment suitable to catalyze the precisesplicing of the target RNA with the 3' "exon" sequences. However, in theabsence of the appropriate target RNA, the ribozyme sequences can stillcatalyze scission at the 3' "exon" junction (similar hydrolysis is seenfor Group I self-splicing intons (Zaug et al., Science 231:470-475(1986)), and may be able to catalyze illegitimate splicing eventsthrough transient base-pairing of the ribozyme with heterologous RNAsequences (which may include their own). Such side-reactions andillegitimate splicing events are unwanted, and may be deleterious. Forexample, if trans-splicing is to be used for conditional delivery of atoxin in vivo, illegitimate trans-splicing might result in unexpectedexpression of the toxic activity. Spontaneous cleavage at the 3' "exon"junction would lower the efficiency of trans-splicing.

To help avoid these problems, "pro-ribozyme" forms of the trans-splicingRNAs have been constructed wherein for example, helix P8 is disrupted.The pro-ribozymes are constructed to contain extra self-complementarysequences which cause the catalytic center of the ribozyme to bemis-folded. The pro-ribozymes are inactive in the absence of theintended target RNA; active forms are only formed after base-pairing ofthe ribozyme and target RNAs--with consequent displacement of theinterfering secondary structure within the ribozyme. Pro-ribozymes areintended to be catalytically inert species in the absence of the targetRNA, to eliminate unwanted self-cleavage, self-splicing and illegitimatetrans-splicing reactions in vitro and in vivo (FIG. 12).

The pro-ribozymes described here are conformationally disrupted andtherefore inactive forms of the trans-splicing activities. Thus thepro-ribozymes possess little self-cleavage activity. They are onlyre-activated by specific interaction with the target RNA, and thus aresubstrate-activated ribozymes which are less likely to catalyzetrans-splicing to an unintended target RNA. Trans-splicing ribozymes areintended to be used for the delivery of new gene activities in vivo, andany reduction in the extent of unwanted side reactions or illegitimatesplicing is desirable, and may be necessary.

While the disruption of helix P8 has been exemplified here for thetrans-splicing pro-ribozymes, other helices which are required forcatalytic activity could also have been used.

The same approach, of disrupting the conformation of a catalyticallyimportant structure in such a way that only base-pairing with theintended substrate RNA will allow the formation of an active ribozyme,could be applied to other ribozyme designs. For example, the loopsequence of a "hammerhead" type endoribonuclease (Haseloff et al.,Nature 334:585-591 (1988)) could be extended and made complementary toone of the "anti-sense" arms of the ribozyme--similar to the abovemodification of helix P8. Endoribonuclease activity would only beexhibited after base-pairing with the chosen target RNA, displacement ofthe disrupting secondary structure, and reformation of the stem-loopstructure required for catalysis. This would effectively increase thespecificity of the ribozyme of its target.

In addition, the activation of a pro-ribozyme need not rely onbase-pairing with the substrate itself. Instead, a chosen third RNA orssDNA or even protein might be required for activity. An additionalbase-pairing or RNA-protein interaction would be required for theformation of an active ribozyme complex. The availability of suchadditional components would determine ribozyme activity, and could beused to alter ribozyme selectivity.

The ribozyme or pro-ribozyme of the invention may be introduced into anyhost cell, prokaryotic or eukaryotic and especially into a plant ormammalian host cell, and especially a human cell, either in culture orin vivo, using techniques known in the art appropriate to such hosts.The ribozymes of the invention may also be engineered to destroyviruses. In one embodiment, the ribozyme or pro-ribozyme of theinvention is provided in a genetically stable manner to a host cellprior to a viral attack. Infection by the appropriate virus, orexpression of the latent virus in such host cell, (resulting in theappearance of the ribozyme's or pro-ribozyme target RNA in thehost-cell), would stimulate the catalytic activity of the ribozyme anddestruction of the viral RNA target and/or production of a toxin viatrans-splicing resulting in death of the virus infected cells. Inanother embodiment, the ribozyme or pro-ribozyme may be engineered andpackaged into the virus itself. Such embodiments would be especiallyuseful in the design of viruses for investigative purposes, wherein theribozyme or pro-ribozyme may be designed to destroy the function of aspecific viral RNA and thus allow the study of viral function in theabsence of such RNA. Viruses carrying ribozymes may also be used ascarriers to transfect host cells with a desired ribozyme or pro-ribozymeactivity.

Male or female sterility may be engineered in agronomically importantspecies using the ribozymes or pro-ribozymes of the invention. Forexample, male sterility in tobacco may be engineered by targetting TA29or TA13 mRNA (tobacco anther-specific genes; Seurinck, J. et al., Nucl.Acids Res. 18:3403 (1990) with a ribozyme or pro-ribozyme of theinvention that trans-splices the DTA 3' exon into those targets.

The form of crop plants may be manipulated by selective destruction ormodification of tissues using the ribozymes or pro-ribozymes of theinventions. For example, seedless fruits may be made by targetting theseed storage protein mRNA with a ribozyme or pro-ribozyme of theinvention that trans-splices the DTA 3' exon into the target.

Transgenic plants may be protected against infection by expression ofvirus-specific ribozymes or pro-ribozyme to kill infected cells. Thiswould be an artificial form the "hypersensitive response." For example,cucumber mosaic virus coat protein mRNA may be targeted with a ribozymeor pro-ribozyme of the invention that trans-splices the DTA 3' exon intothe target.

Populations of micro-organisms may be made resistant to specificpathogens by introduction of trans-splicing ribozymes or pro-ribozymes.For example, cheese-making bacteria may be made resistant to phageinfection by targetting the phage RNA with a bacterial toxin gene orlytic enzyme encoded by the 3' exon provided by the ribozyme orpro-ribozyme of the invention, for example, which would interfere withphage replication by causing premature lysis after phage infection.

Virus pathogens could be constructed to deliver toxic activities viatrans-splicing. In this way, specific cell types could be targeted forablation, such as for cancer or viral therapy. For example, HIV mRNA maybe targeted by a ribozyme or pro-ribozyme of the invention that carriesthe DTA 3' exon, for either virus or liposome-delivery.

The examples below are for illustrative purposes only and are not deemedto limit the scope of the invention.

EXAMPLES Example 1 Construction and Characterization of a CAT-LacZTrans-Splicing Ribozyme

I. PCR Amplification and Cloning of the Ribozyme of the Invention

Following the guidelines outlined above, a trans-splicing fusionribozyme was designed that will splice a portion of the amino-terminalcoding sequence of E. coli β-galactosidase (LacZ) mRNA to a site in thechloramphenicol acetyl transferase (CAT) mRNA (FIG. 3). The sections ofnew sequence flanking the T. thermophila ribozyme core and the 3' exonwere synthesized as oligonucleotides. The intact ribozyme sequence wasthen assembled by successive polymerase chain reactions, using thesynthetic adaptor oligonucleotides as primers with ribozyme andβ-galactosidase DNA templates (while there are other methods available,this method is most convenient).

For the construction of a ribozyme capable of splicing β-galactosidase(LacZ) α-peptide coding sequence to a site in the 5' coding sequence ofthe chloramphenicol acetyl transferase (CAT), three oligonucleotideswere synthesized.

    __________________________________________________________________________    Oligonucleotide 1    5'-GGCCA AGCTT CTTTA CGATG CCATT GGGAT ATATC AACGG    TGGTA TAAAC CCGTG GTTTT TAAAA GTTAT CAGGC ATGCA CC-3'     SEQ ID NO. 2!    Oligonucleotide 2    5'-GATTA GTTTT GGAGT ACTCG TACGG ATTCA CGGCC GTCGT    TTTAC AA-3'  SEQ ID NO. 3!    Oligonucleotide 3    5'-GGCGG AATTC TTACA ATTTC CATTC AGGCT GCGCA ACTGT TGG-    3'  SEQ ID NO. 4!    __________________________________________________________________________

Oligonucleotides 2 and 3 (200 pmoles each) were combined with 0.1 μgPvuII-cut pGEM4 DNA (which contained the LacZ α-peptide sequence), andsubjected to PCR amplification in a volume of 100 μl containing:

50 nm KCl,

10 mM Tris-HCl pH 8.3,

1.5 mM MgCl₂,

0.4 mM dNTPs,

0.1% gelatin, and

5 U TaqI DNA polynerase,

and incubated for 30 cycles, 1 min @ 94° C., 2 mins @ 50° C., 2 mins @72° C.

Plasmid pGEM4 is commercially available from Promega Corporation,Madison Wis., USA.

The amplified product of 210 base-pairs was purified using low-gellingtemperature agarose electrophoresis, and was used as primer in a secondround of PCR amplification.

Following the second round of PCR amplification, 2.0 μg of 210 base-pairamplified product, 200 pmoles oligonucleotide 1 and 0.1 μg 450 base-pairfragment containing the T. thermophila IVS were mixed and subjected toPCR amplification using the conditions shown above. The resulting 660base-pair product was digested with the restriction endonucleases EcoRIand HindIII, and cloned into the plasmid vector pGEM4. The completesequence of the CAT-LacZ α-peptide ribozyme DNA sequence is presented asSEQ ID NO. 5 and FIG. 3B.

The cloning vector containing the cloned sequences was transformed into,and propagated in, the bacterial host XL1/Blue (Strategene, La Jolla,Calif.), using techniques known in the art (Maniatis, Molecular Cloning,A Laboratory Guide, 2nd edition, 1989, Cold Spring Harbor Laboratory,Publishers). However, any bacterial host capable of stably maintainingthe vector may be used, for example the JM109.

The plasmid may be extracted from the host cell for further analysisusing techniques commonly known in the art (Maniatis, Molecular Cloning,A Laboratory Guide, 2nd edition, 1989, Cold Spring Harbor Laboratory,Publishers).

II. In vitro Transcription of Cloned Ribozyme and Target RNAs

Using standard procedures, cloned sequences were purified from thebacterial host and the plasmid linearized using a restrictionendonuclease that does not cut the ribozyme sequence, (for example,EcoRI), and transcribed using T7 RNA polymerase in a volume of 100 μl,containing:

5 μg linearized plasmid DNA,

40 mM Tris-HC pH 7.5,

6 mM MgCl₂,

2 mM spermidine,

10 mM NaCl,

10 mM DTT,

1 mM NTPs (containing 20 μCi α-³² P!UTP, if labelled RNA transcriptswere desired),

100 U RNasin, and

50 U T7 RNA polymerase,

and the reaction was incubated at 37° C. for 2 hours.

RNA transcripts were purified by 5% polyacrylamide gel electrophoresisbefore use (TBE, 7M urea gel). RNAs containing active T.thermophila IVAsequences undergo some spontaneous scission at the 3' intron-exonjunction during transcription. Fragments are removed by electrophoreticpurification for clarity of analysis during subsequent trans-splicingassays.

III. In Vitro Trans-splicing Reaction Conditions

Target and/or trans-splicing ribozymes are incubated under the followingconditions:

0.1-0.5 μg RNA component (amount depends on type of experiment, usuallyribozyme in 5-fold excess of target),

30 mM Tris-HCl pH 7.5,

100 mM NaCl,

2 mM GTP,

5 MM mgCl₂,

in a volume of 5 μl at 42° C., 60 mins.

The reaction is diluted with 95 μl 0.1 mM Na₂ EDTA, 200 mM NaCl, andethanol precipated. The RNAs are then analysed on 5% polyacrylamide gelscontaining TBE buffer, 7M urea and 25% formamide, and autoradiographed.

IV. Assay of Endonucleolytic Activity

After base-pairing of the ribozyme and target, the first step intrans-splicing is the guanosine mediated cleavage of the target RNA atthe intended 5' splice site. Annealing and trans-splicing may beperformed in a buffer such as 30 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mMMgCl₂, 2 mM GTP at 42° C. As the 3' splice site is dispensable for thisreaction, truncated trans-splicing ribozymes should behave ashighly-specific endoribonucleases. To test this activity, shortened invitro transcripts of the CAT-LacZ α-peptide trans-splicing ribozymedescribed above (SEQ ID NO. 5 and FIG. 3) were incubated with CAT mRNAsequences. The CAT-LacZ ribozyme cassette is on a HindIII-EcoRIfragment. The ScaI cleavage site marks a position 5 bases upstream ofthe 3' splice site. The ribozyme specifically cleaved the target RNA atthe expected single site to produce the expected size fragments.

V. The Trans-splicing Reaction

To confirm the ability of the CAT-LacZ α-peptide ribozyme to catalyzethe ligation of 3' exon sequences at the 5' splice site, various formswere incubated with radiolabelled CAT RNA. Ribozyme transcripts weresynthesized from DNA templates which had been 3' truncated at one ofseveral positions, ranging from the end of the ribozyme core through theexon sequence. Incubation with labelled CAT led to the formation of theexpected spliced products, which differed in length depending on theextent of 3' exon sequence.

In addition, a certain proportion of the CAT-LacZ αpeptide ribozymemolecules underwent spontaneous cleavage at the 3' splice site during invitro transcription, similar to the intact T. thermophila intron. Thesecleaved forms, terminated at the guanosine residue adjacent the 3'splice site, were also incubated with CAT RNA. In this case, theribozyme itself is ligated to a 3' portion of the CAT RNA, to produce aproduct of about 550 nucleotides in size. This reaction is similar tothe self-circularization of the intact intron, and the same ligationproduct is found in the other trans-splicing reactions.

VI. Accuracy of the Trans-splicing

The products from a CAT-LacZ α-peptide trans-splicing reaction werereverse-transcribed, and amplified by polymerase chain reaction usingtwo oligonucleotides complementary to sequences on either side of thepredicted splice sites. Amplified sequences were cloned and sequenced.Individual recombinants showed no variation from the expected sequenceof the spliced products. As found in studies with the intact intron,splicing appears to be highly accurate.

Accordingly, the studies above show that a trans-splicing ribozymedesigned according to the guidelines of the invention is capable ofaccurate, effective trans-splicing in vitro.

Example 2 Design of a Trans-Splicing Ribozyme that Provides Plant VirusResistance

Cucumber mosaic virus (CMV) is a pandemic virus with a large number ofknown strains. Nine sequence strains are shown in the region of thestart of their coat protein cistron encoded in RNA 3 and the subgenomicmRNA 4 (SEQ ID NOS. 7-25; FIGS. 4(A) and 5). Two sites have been chosenwhich are conserved in sequence and downstream from the AUG start codonof the coat-protein. Oligonucleotides for the construction of ribozymescapable of trans-splicing the ile-mutant form of DTA into the CMV coatprotein mRNA are shown in FIG. 4B and is discussed below.

The trans-splicing ribozymes shown in FIG. 4C are targetted to the CMVvirus sequences shown in FIG. 4B and will result not only in thecleavage of the CMV RNA molecules but in the expression of diphtheriatoxin A-chain in the infected cell. The trans-splicing cassettes shownin FIG. 4 may be transformed into any CMV-susceptible plant speciesusing techniques known in the art, and transgenic progeny challenged byCMV infection. The design of the ribozyme is such that virus infectionis necessary to initiate toxin production via RNA trans-splicing becausethe ribozyme itself is not translated. The localized death of theinfected cells that results from expression of the toxin could limitreplication and spread of the virus within the plant giving anartificial hypersensitive response.

Example 3 Construction and Characterization of a Gal4-Diphtheria Toxin AChain Trans-Splicina Ribozyme

According to the invention and the methods described in Example 1, afusion ribozyme has been designed that is a Gal4-Diphtheria toxin Achain trans-splicing ribozyme (FIG. 6). The sequence of this ribozyme isshown as SEQ ID NO. 6. The GAL4-DTA ribozyme cassette is a SalI-XhoIfragment. The ScaI site marks a position 5 bases upstream of the 3'splice site. This ribozyme is capable of splicing the coding sequencefor the A chain of the diphtheria toxin to a site in the 5' region ofthe GAL4 mRNA. This trans-splicing activity is active both in vitro (asabove) and in vivo (below). The major criteria for successful design ofthe GAL4-DTA ribozyme, and any trans-splicing ribozyme thattrans-splices a sequence encoding a toxic product, are not only theefficient and precise catalysis of trans-splicing, but also thatexpression of the toxic product, for example, DTA does not occur in theabsence of trans-splicing.

The catalytic portion of the ribozyme is constructed according to thedesign outlined above, and 5' and 3' splice sites chosen within the 5'coding regions of GAL4 and DTA, respectively. The 3' exon sequencecorresponds to that of a DTA gene already used for expression ineukaryotes, except for the removal of the first AUG codon and severalproximal amino acids. The original C. diphtheriae form of DTA alsodiffers in this 5' region, utilizing a CUG codon for translationinitiation. The original DTA sequence also contains a signal peptideleader sequence which is absent.

These ribozyme molecules can undergo spontaneous scission at the 3'splice site. Given the extreme toxicity of DTA, it is important that anyliberated 3' exon sequences not give rise to toxic translation products.The 3' exon contained an in-frame methionine at position 13, which couldconceivably give rise to a truncated but toxic polypeptide. To eliminatethis possibility, the wild-type sequence (Rz-DTA_(met)) was altered frommethionine at this position to isoleucine (Rz-DTA_(ile)) or leucine(Rz-DTA_(leu)) in two separate ribozyme constructions (FIG. 6).

Example 4 In Vivo Activity of the Ribozymes of the Invention

I. Introduction

The in vivo activity of a ribozyme designed according to the guidelinesprovided herein, and the ability of such a ribozyme to deliver new geneactivities to host cells, was demonstrated using the Gal4-Diphtheriatoxin A chain trans-splicing ribozyme described (Example 3 and in FIG.6) to deliver the highly toxic diphtheria toxin A product to a hostcell. In this system, Drosophila was the chosen host and it was desiredto control expression of the ribozyme of the invention in atissue-specific manner within the Drosophila host.

Diphtheria toxin is secreted by Corynebacterium diphtheriae lysogenicfor B phage. The toxin is produced as a single polypeptide whichundergoes proteolysis to produce A and B chains. The A chain (DTA)contains a potent ADP ribosylase activity which is specific for theeukaryote translation elongation factor EF-2. The presence of even a fewmolecules of this enzyme is enough to cause cessation of translation andeventual death in a variety of eukaryote cells. The B chain allowsintracellular delivery by attachment of the toxin to cell surfacereceptors by binding mannose residues, is endocytosed and enters thecytoplasm by vesicular fusion.

In the absence of the B-chain, the A-chain is much less toxic whenpresent extracellularly. This property, and its extreme toxicity, havesuggested its use for ectopic ablation experiments. For example,sequences encoding DTA have been expressed in transgenic mice, using anopsin promoter to drive expression in developing eyes. The resultingmice are blind, with deformed eyes (Breitman, M. L., Science238:1563-1565 (1987)). In other studies, ablation of the mouse pancreaswas performed (Palmiter, R. D. et al., Cell 50:435-443 (1987)) and Wert,S. E. et al., Am. Rev. Respir. Dis. 141 (no. 4, part 2):A695 (1990)described ablation of alveolar cells by use of a chimeric geneconsisting of the promoter and 5' flanking sequence of the humansurfactant protein C gene (expressed in type II alveolar cells) and theDTA gene.

However, using this type of approach, it is not possible to maintain orpropagate transformed organisms which might have more severe, or lethalphenotypes. In addition, transformation of certain species, such asDrosophila, with intact DTA sequences has not been reported to date.Leaky expression of the DTA gene during such transformations leads toimmediate death.

II. The Drosphila System

A general method for targeting gene expression in Drosophila has beendeveloped. First, the system allows the rapid generation of individualstrains in which ectopic gene expression can be directed to differenttissues or cell types: the enhancer detector technique is utilized(O'Kane, C. J. and Gehring, W. J., Proc. Natl. Acad. Sci. USA: 9123-9127(1987); Bellen et al., Genes and Development 3:1288-1300 (1989); Bier etal., Genes and Development 3:1273-1287 (1989)) to express atranscriptional activator protein in a wide variety of patterns inembryos, in larvae and in adults. Second, the method separates theactivator from its target gene in distinct lines, to ensure that theindividual parent lines are viable: in one line the activator protein ispresent but has no target gene to activate, in the second line thetarget gene is silent. When the two lines are crossed, the target geneis turned on only in the progeny of the cross, allowing dominantphenotypes (including lethality) to be conveniently studied.

To ectopically express only the gene of interest, a transcriptionalactivator that has no endogenous targets in flies is required. Anactivator from yeast, Gal4, can activate transcription in flies but onlyfrom promoters that bear Gal4 binding sites (Fischer et al., Nature332:853-865 (1988)). To target gene expression, Gal4 is restricted toparticular cells in two ways: either Gal4 transcription is driven bycharacterized fly promoters, or an enhancerless Gal4 gene is randomlyintegrated in the Drosophila genome, bringing it under the control of adiverse array of genomic enhancers. To assay transactivation by Gal4,flies that express Gal4 are crossed to those bearing a lacZ gene whosetranscription is driven by Gal4 binding sites (Fischer et al., Nature332:853-865 (1988)). β-galactosidase is expressed only in those cells inwhich Gal4 is first expressed. Tissue- and cell-specific transactivationof lacZ has been demonstrated in strains in which Gal4 is expressed andin which a variety of patterns are established.

With this system, it is now possible: 1) to place Gal4 binding sitesupstream of any coding sequence; 2) to activate that gene only withincells where Gal4 is expressed and 3) to observe the effect of thisaberrant expression on development. In cases where ectopic expression islethal, this method allows the two parent lines (one expressing Gal4,the other carrying a silent gene bearing Gal4 binding sites in itspromoter) to be stably propagated. Phenotypes can then be studied in theprogeny of a cross.

III. Vectors

The vectors utilized as starting materials in these studies include:

1) PGATB and pGATN (FIG. 9): These vectors are used for cloningpromoters and enhancers upstream of a promoterless Gal4 gene.

Vectors were constructed in which either a unique NotI or BamHI site isinserted upstream of the Gal4 coding region. Once a promoter has beenlinked to the Gal4 coding sequence, the gene can be excised from thepHSREM vector backbone (Knipple and Marsella-Herrick, Nucl. Acids Res.16:7748 (1988)) and moved into a P-element vector. The Rh2 promoter hasbeen cloned (Mismer et al., Genetics 120:173-380 (1988)) into thisvector and flies have been generated in which Gal4 is expressed only inthe ocelli.

2) pGawB: This is a Gal4 vector for use in enhancer detection.

An enhancerless Gal4 gene was subcloned into the vector plwB (Wilson etal., Genes and Development 3:1301-1313 (1989)) to create pGawB. plwB wasfirst digested with HindIII to remove the lacZ gene and the N-terminusof the P-transposase gene. These were replaced with the entire Gal4coding region behind the TATA box of the P-transposase gene.

3) pUAST (FIG. 10): This plasmid was used for cloning coding sequencesdownstream of the Gal UAS.

A vector into which genes can be subcloned behind the Gal4 UAS (UpstreamActivation Sequence) was constructed in the P-element vector, pCaSpeR3(C. Thummel, Univ. of Utah Medical Center, Salt Lake City, Utah,personal communication). Five Gal4 binding sites were inserted, followedby the hsp70 TATA box and transcriptional start, a polylinker, and theSV40 intron and polyadenylation site. Unique sites into which genes, orcDNAs, can be inserted include: EcoRI, BGlII, NotI, XhoI, KpnI and XbaI.

IV. Drosophila Strains

The genetic techniques described herein used to characterize the strainsof Drosophila utilized in these studies are well known in the art("Genetic Variations of Drosophila melanogaster," D. Lindsley and E. H.Grell, eds).

The P-element transposons are mobilized using the "jumpstarter" strainthat carries Δ2-3, a defective P-element on the third chromosome thatexpresses high levels of a constitutively active transposase (Robertsonet al., Genetics 118:451-470 (1988)). The three stocks currently used togenerate and map the insertion lines were deposited in the DrosophilaStock Center, Indiana University Department of Biology, Jordan Hall A503, Bloomington, Ind. 47405:

1: y w; +/+; Sb P ry⁺, Δ2-3!/TM6, Ubx

2: w; +/+; TM3, Sb/CxD (deposit no. 3665)

3: w; CyO/Sco; +/+(deposit no. 3666)

where the genetic characteristics of the three chromosomes are separatedby semicolons. Thus, for example, in strain 1, the first chromosome (theX chromosome) is homozygous for yellow and white ("y w"), the secondchromosome is wild-type ("+/+"), and the third chromosome carries thestubble gene ("Sb"), and the P element transposon rosy gene ("ry⁺ ") andΔ2-3, while the second third chromosome carries balancer inversions("/TM6, Ubx").

V. Strateqy for Generating Gal4 Expression Patterns

A. Scheme used to isolate transformants

    ______________________________________    Constructs are injected into embryos derived from the stock;    ♀♀ y w/y w; Δ2-3, Sb/TM6, Ubx                     X     ♂♂ y w/Y; Δ2-3, Sb/TM6, Ubx    F1; Establish single lines    ♀ y w/y w; Δ2-3, Sb/TM6, Ubx                     X     ♂ y w/Y; +/+                     or    ♀ y w/Y; Δ2-3, Sb/TM6, Ubx                     X     ♂ y w/y w; +/+    F1; Select  w±! and  Sb±! progeny and establish stocks    ♀ y w/y w; +/TM6, Ubx                     X     ♂ y w/Y; +/+                     or    ♀ y w/Y; +/TM6, Ubx                     X     ♂ y w/y w; +/+    ______________________________________

B. Schemes used to iump the enhancerless Gal4 insert

    __________________________________________________________________________    1. Jumps from the X-chromosome    ♀♀ FM3/FM7, w; +/+                       X ♂♂ y w/Y; Δ2-3, Sb/TM6, Ubx    ♀♀ FM7, w/ P Gal4, w.sup.+ !                       X ♂♂FM7/YU; Δ2-3, Sb/+    ♀♀ FM7, w/ P Gal4, w.sup.+ !; Δ2-3,                       X ♂♂ y w/Y; +/+    ♀ FM7, w/ y w; Δ2-3, Sb/+                       X ♂ y w/Y; +/+    Select  w.sup.+ ! and  B! progeny and establish stocks    2. Jumps from the Δ2-3-chromosome    ♀♀ y w/y w                       X ♂♂ y w/Y; P Gal4, 2.sup.+ !, Δ2-3,                       Sb/+    Select  w.sup.+ ! and  Sb.sup.+ ! progeny and establish    __________________________________________________________________________    stocks.

C. Chromosomal segregation

To analyze the segregation of the insertions two stocks are used:

w;+/+; TM3, Sb/CxD and w; CyO/Sco;+/+.

Method

To create a large number of strains that express Gal4 in a cell- ortissue-specific manner enhancer detection vectors have been built thatcarry different versions of the Gal4 gene. Two genes, encoding eitherthe full-length protein or a truncated protein, have been cloned intorosy (ry⁺) and white (w⁺) P-element vectors (modified versions of plArBand plwB; Wilson et al., Genes and Development 3:1301-1313 (1989)).Using ry⁺ or w⁺ as a screen, these vectors have been mobilized byintroduction of the Δ2-3 gene (Robertson et al., Genetics 118:461-470(1988)). To visualize the expression pattern of Gal4, the Gal4 insertionlines are crossed to a strain that carries the lacZ gene under thecontrol of the Gal4 UAS (Fischer et al. Nature 332:853-865 1988).Embryos, larvae and adults derived from these crosses are screened forβ-galactosidase expression either by an enzyme assay, with X-gal as asubstrate, or by staining with monoclonal antibodies againstβ-galactosidase. β-galactosidase encoded by the UAS-lacZ construct islocalized in the cytoplasm.

Approximately 500 Gal4-insertion strains have been screened and manythat can be used to activate genes in specific tissues have beenidentified such as, for example, epidermal stripes, mesoderm, thecentral nervous system and the peripheral nervous system. Many of thelines express β-galactosidase in the salivary glands as well as in othertissues. It is possible that in constructing the enhancerless-Gal4transposon a position-dependent salivary gland enhancer was fortuitouslygenerated.

VI. Sample Screen

    ______________________________________    # of Strains            No Staining                       Salivary Gland                                   Other Tissues                                            %    ______________________________________    9       +          -           -        5.8    45      -          +           -        28.8    81      -          +           +        51.9    21      -          -           +        13.5    156                                     100.0    ______________________________________

To activate a gene (Gene X) in a specific pattern, a Gal4 insertion lineis selected and crossed to a strain that carries Gene X cloned behindthe GAL UAS.

VII. Summary of the GAL4/UAS System without the Ribozyme

The Gal4/UAS system is a two-part system for controlling geneactivation. The method is versatile, can be tissue-specific and does notappear to exhibit a basal level of expression except perhaps, asdescribed herein, for a UAS-DTA construct. It can be used to ectopicallyexpress characterized genes, to express modified genes that wouldotherwise be lethal to the organism and to express genes from otherspecies to study their effect on Drosophila development. Since themethod makes it possible to produce dominant, gain-of-functionmutations, epistasis tests and screens for enhancers or suppressors ofvisible or lethal phenotypes can be carried out. The Gal4 system alsoallows the expression of toxic products to study the consequences ofcell- and tissue-specific ablation.

VIII. Use of Gal4-Expressing Drosophila with the DTA Ribozyme of theInvention

Expression of the fusion ribozyme carrying the sequences encoding theDTA protein was placed under the control of a the GAL4 UAS (upstreamactivator sequence) in PUAST (FIG. 10). As stated supra, using modifiedP-element enhancer-trap vectors described above, a large number ofstable lines of Drosophila were constructed which each express the yeasttranscriptional activator GAL4 in specific spatial and temporal patternsin the developing flies. Any gene under the control of the GAL4 upstreamactivator sequence (UAS) can be transformed and maintained singly, theninduced in particular Drosophila tissues by genetic crossing to lineswhich express GAL4 (FIG. 7). However, it was not possible to takeadvantage of the Gal4 system for expression of DTA per se withoutfurther modification, due to the difficulty in producing UAS-DTAtransformants through leaky expression of the DTA.

It was found that use of this two-element system as a means ofconditionally expressing DTA via a trans-splicing ribozyme (FIG. 6)overcame these problems. In those cells expressing GAL4, the GAL4protein provides the activity necessary for ribozyme transcription, andthe GAL4 mRNA provides the target for trans-splicing necessary for DTAproduction.

Drosophila embryos may be injected with ribozyme sequences placed underthe control of a UAS promoter as described above, using techniques knownin the art. Embryos injected with the Rz-DTA_(met) construction will notsurvive, whereas normal transformed flies were obtained from embryosinjected with both Rz-DTA_(ile) and Rz-DTA_(leu). This result suggestedthat the internal AUG codon was indeed acting as an initiation codon forthe translation of a toxic product after injection. The codon isadjacent to proposed NAD+ binding site in the DTA sequence, and tosequences conserved in the distantly related exotoxin A, another EF-2specific ADP-ribosylase from Pseudomonas aeruginosa.

Transgenic flies containing the Rz-DTA_(ile) and Rz-DTA_(leu) sequencesunder control of the GAL4 UAS were crossed to flies producing GAL4 inparticular patterns of expression. For example, in one characterizedline, line 1J3, the GAL4 gene was been inserted near the hairy gene, andmirrored its pattern of expression. The hairy gene product is producedin epidermal stripes in the even-numbered abdominal segments duringembryogenesis. When a UAS-driven LacZ gene was introduced into 1J3 inwhich GAL4 is expressed in the same pattern as the hairy gene product,β-galactosidase was found localized within the even-numbered stripes.When flies containing. the Rz-DTA_(leu) gene were crossed to thisGAL4-expressing line, normal progeny resulted. However, when fliescontaining Rz-DTA_(ile) were crossed to the GAL4-expressing line,development of the progeny was arrested in embryogenesis. Darker coloredbands were evident on the cuticles of the embryos, consistent with thedeath of underlying cells. When cuticle preparations were examined, theeven-numbered denticle bands were disrupted or missing, particularlythose of the 4th, 6th and 8th stripes (FIG. 11). Other specific patternsof cell death were observed when the containing Rz-DTA_(ile) flies arecrossed to different GAL4 expressing genes.

Example 5 Design of Pro-ribozymes

As a test for the design of pro-ribozymes, the CAT-LacZ trans-splicingribozyme which described earlier was modified (FIG. 2). Phylogeneticcomparisons and mutational analysis (for review, see Cech, Ann Rev.Biochem. 59:543-568 (1990)) have, indicated that a core region of thegroup I self-splicing introns is highly conserved and important foractivity (FIG. 8). For the construction of trans-splicing pro-ribozymesa helix immediately adjacent to this region, P8, was disrupted. In thefirst experiments, 13 or 18 nucleotides of new sequence were introducedinto the 5' strand and loop of helix P8, to produce pro-ribozyme 1 and2, respectively. The extra nucleotides were complementary to the 5'"anti-sense" portion of the ribozyme, while the flanking sequences wereadjusted to conserve (1) the actual sequences at the base of P8, and (2)the extent of base-pairing possible within P8 (FIG. 13). The extent ofself-complementarity between the sequences inserted into helix P8 andthe 5' "anti-sense" region of the pro-ribozyme is such that this newhelix would be expected to form in nascent transcripts, in preference tohelix P8. The formation of this alternative helix would also be expectedto disrupt flanking secondary and perhaps tertiary interactions withinthe catalytic core of the ribozyme. Thus, mis-folding of thepro-ribozyme would render it catalytically inactive (FIG. 14). However,base-pairing of the pro-ribozyme with the intended target RNA woulddisplace the P8-"anti-sense" base-pairing, sequester the "anti-sense"sequences and-allow re-formation of the P8 helix and an active catalyticdomain. Displacement of the P8-"anti-sense" helix results in a greatersum of base-pairs and allows proper folding of the catalytic domain, soshould be energetically favored.

CAT-LacZ pro-ribozymes

Cloned sequences corresponding to the two CAT-LacZ pro-ribozymes wereconstructed using PCR-mutagenesis as discussed above, and RNAs wereproduced by in vitro transcription. The CAT-LacZ trans-splicing ribozymewas observed to undergo scission during transcription at the 3' splicejunction, as a result of hydrolysis catalyzed by the intron sequences.Similar hydrolysis is seen in in vitro transcripts of the unmodifiedTetrahymena thermophila intron. In contrast, transcripts of thedifferent CAT-LacZ pro-ribozymes are more stable, with little cleavageevident under the same conditions (FIG. 15). This indicates that thepro-ribozymes are inactive, which would be expected if the catalyticsequences were mis-folded. Truncated forms of the pro-ribozymes weretested for specific endoribonuclease activity directed against the CATRNA. CAT-LacZ pro-ribozyme RNAs were transcribed from templatestruncated at the ScaI site, to remove the 3' splice junction and LacZsequences. Both ribozyme and pro-ribozyme RNAs are stable after removalof the 3' splice site. Incubation of the truncated pro-ribozymes withCAT RNA led to specific cleavage of the target RNA to give fragments ofthe expected sizes (FIG. 16). Specific cleavage activity was seen at 37,45 and 50 degrees.

Pro-ribozyme forms of the GAL4-DTA trans-splicing ribozyme were alsoconstructed (FIG. 17). Regions of 20 nucleotides (complementary to the"anti-sense" region) were inserted into the 5' strand and loop of helixP8. The two pro-ribozymes differed in the extent of base-pairingpossible in the modified helices P8, and GAL4-DTA pro-ribozyme 1possessing both a longer stem and fewer (3) accessible bases in theloop. The helix P8 of GAL4-DTA pro-ribozyme 2 more closely resemblesthat of the CAT-LacZ pro-ribozyme 2, with a larger loop (14 bases)containing sequences complementary to the "anti-sense" region.Transcripts of the GAL4-DTA pro-ribozymes are more stable than those ofthe unmodified ribozyme. In particular, pro-ribozyme 2 is mainly intactafter incubation in conditions that result in essentially completeself-cleavage of the ribozyme form (30'@50° C., 10 mM MgCl₂, 2 mM GTP,see FIG. 18).

Having now fully described the invention, it will be understood by thosewith skill in the art that the scope may be performed within a wide andequivalent range of conditions, parameters and the like, withoutaffecting the spirit or scope of the invention or any embodimentthereof.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 56    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 517 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    TGACGCAATTCAACCAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTAAATAGCA60    ATATTTACCTTTGGAGGGAAAAGTTATCAGGCATGCACCTCCTAGCTAGTCTTTAAACCA120    ATAGATTGCATCGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCG180    TTCAGTACCAAGTCTCAGGGGAAACTTTGACATGGCCTTGCAAAGGGTATGGTAATAAGC240    TGACGGACATGGTCCTAACCACGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATAT300    GGATGCAGTTCACAGACTAAATGTCGGTCGGGGAAGATGTATTCTTCTCATAAGATATAG360    TCGGACCTCTCCTTAATGGGAGGTAGCGGATGAATGGATGCAACACTGGAGCCGCTGGGA420    ACTAATTTGTATGCGAAAGTATATTGATTAGTTTTGGAGTACTCGTAAGGTAGCCAAATG480    CCTCGTCATCTAATTAGTGACGCGCATGAATGGATTA517    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 82 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    GGCCAAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAACCCGTGGTTTT60    TAAAAGTTATCAGGCATGCACC82    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 47 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GATTAGTTTTGGAGTACTCGTACGGATTCACGGCCGTCGTTTTACAA47    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    GGCCGAATTCTTACAATTTCCATTCAGGCTGCGCAACTGTTGG43    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 623 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GGGAGACCGGAAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAAGCCGT60    GGTTTTTAAAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCAT120    CGGTTTAAAAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAA180    GTCTCAGGGGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATG240    GTCCTAACCACGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTTC300    ACAGACTAAATGTCGGTCGGGGAAGATGTATTCTTCTCATAAGATATAGTCGGACCTCTC360    CTTAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTA420    TGCGAAAGTATATTGATTAGTTTTGGAGTACTCGTACGGATTCACTGGCCGTCGTTTTAC480    AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCC540    CTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGC600    GCAGCCTGAATGGAAATTGTAAG623    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1038 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GTCGACCTTTTTAAGTCGGCAAATATCGCATGTTTGTTCGATAGACATCGAGTGGCTTCA60    AAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAA120    AAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGG180    GGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAAC240    CACGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTTCACAGACTA300    AATGTCGGTCGGGGAAGATGTATTCTTCTCATAAGATATAGTCGGACCTCTCCTTAATGG360    GAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAG420    TATATTGATTAGTTTTGGAGTACTCGTCTCGATGATGTTGTTGATTCTTCTAAATCTTTT480    GTGATTGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAA540    AAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGG600    TTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCG660    CTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTC720    GCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAA780    CCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGTGCTTCG840    CGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAAC900    TGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAA960    CGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGG1020    CGATCTTTGTGACTCGAG1038    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 134 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GTTTAGTTGTTCACCTGAGTCGTGTGTTTTGTATTTTGCGTCTTAGTGTGCCTATGGACA60    AATCTGGATCTCCCAATGCTAGTAGAACCTCCCGGCGTCGTCGCCCGCGTAGAGGTTCTC120    GGTCCGCTTCTGGT134    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 134 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    GTTTAGTTGTTCACCTGAGTCGTGTTTTCTTTGTTTTGCGTCTCAGTGTGCCTATGGACA60    AATCTGGATCTCCCAATGCTAGTAGAACCTCCCGGCGTCGTCGCCCGCGTAGAGGTTCTC120    GGTCCGCTTCTGGT134    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    GTTATTGTCTACTGACTATATAGAGAGTGTTTGTGCTGTGTTTTCTCTTTTGTGTCGTAG60    AATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGTCGTAACCGTCGACGTC120    GTCCGCGTCGTGGTTCCCGCTCCGCCCCCTCC152    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    GTTATTGTCTACTGACTATATAGAGAGTGTGTGTGCTGTGTTTTCTCTTTTGTGTCGTAG60    AATTGAGTCGAGTCATGGATAAATCTGAATCAACCAGTGCTGGTCGTAACCGTCGACGTC120    GTCCGCGTCGTGGTTCCCGCTCCGCCTCCTCC152    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 131 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AGAGAGTGTGTGTGCTGTGTTTTCTCTTTTGTGTCGTAGAATTGAGTCGAGTCATGGACA60    AATCTGAATCAACCAGTGCTGGTCGTAACCGTCGACGTCGTCCGCGTCGTGGTTCCCGCT120    CCGCCCCCTCC131    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 154 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GTTATTGTCTACTGATTGTATAAAGAGTGTGTGTGTGCTGTGTTTTCTCTTTTACGTCGT60    AGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGTCGCAACCGTCGACG120    TCGTCCGCGTCGTGGTTCCCGCTCCGCCCCCTCC154    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 154 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GTTATTGTCTACTGACTATATAGAGAGTGTGTGTGTGCTGTGTTTTCTCTTTTGTGTCGT60    AGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGTCGTAACCGTCGACG120    TCGTTTGCGTCGTGGTTCCCGCTCCGCCTCCTCC154    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 130 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    GAGTGTGTATGTGCTGTGTTTTCTCTTTTGTGTCGTAGAATTGAGTCGAGTCATGGACAA60    ATCTGAATCAACCAGTGCTGGTCGTAACCGTCGACGTCGTCCGCGTCGTGGTTCCCGCTC120    CGCCCCCTCC130    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 152 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    GTTATTGTCTACTGACTATATAGAGAGTGTGTGTGCTGTGTTTTCTCTTTTGTGTCGTAG60    AATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGTCGTAACCATCGACGTC120    GTCCGCGTCGTGGTTCCCGCTCCGCCCCCTCC152    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 78 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    GGAGGGGGCGGAGCGGGAACCACGACGCGGACGACGTCGACGGTTACGACCAGCCCTGGT60    AGATTCAGATTTGTCCAT78    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    TTTGCGTCTTAGTGTGCCTATGGACAAATCTGGATCTCCCAATGCTAGT49    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 49 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    TTTGCGTCTCAGTGTGCCTATGGACAAATCTGGATCTCCCAATGCTAGT49    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGATAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    TTTACGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 56 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    TTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGGT56    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    AATTTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGCA59    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    GCACTGGTTGATTCAGATTTGTCCATGACTCGACTCAATTCTACGACACAA51    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    AATTTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGCA59    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    AGCATTGGTATCATCAGGTTTGT23    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GTTGATGATGTTGTTGATTCT21    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    MetAspLysPheAspAspValValAspSer    1510    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    ATGGACAAATTTGATGATGTTGTTGATTCT30    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    AATTTTGTGTCGTAGAATTGAGTCGAGTCATGGACAAATCTGAATCAACCAGTGCTGCA59    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    AGCCATCCTTGGTTCAG17    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    GTAAGGGTGGATGTT15    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    MetAspLysSerGluLeuArgValAspVal    1510    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    ATGGACAAATCTGAATTAAGGGTGGATGTT30    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    TCTCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCA60    CGGGACTAAA70    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    MetGluAsnPheSerSerTyrHisGlyThrLys    1510    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    TCTCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATTGAAAACTTTTCTTCGTACCA60    CGGGACTAAA70    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 70 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    TCTCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGTTGGAAAACTTTTCTTCGTACCA60    CGGGACTAAA70    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 78 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    ATGAAGCTTCTCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCT60    TCGTACCACGGGACTAAA78    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    MetLysLeuLeuAspAspValValAspSerSerLysSerPheValMet    151015    GluAsnPheSerSerTyrHisGlyThrLys    2025    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 41 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATC41    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    MetGluLysLysIleThrAspSerLeuAlaValValLeuGlnArgArg    151015    Asp    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    ATGGAGAAAAAAATTACGGATTCACTGGCCGTCGTTTTACAACGTCGTGAC51    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 40 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTT40    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    MetLysLeuLeuAspAspValValAspSerSerLysSerPheValMet    151015    GluAsnPheSer    20    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 60 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    ATGAAGCTTCTCGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCT60    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 72 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAA60    CATTTTGAGGCA72    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 479 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    AAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAAGCCGTGGTTTTTAAA60    AGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAAAA120    GGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGG180    AAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCA240    CGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTACAGACTAAATG300    TCGGTCGGGGAAGATGTATTCTTCTCATAACATATAGTCGGACCTCTCCTTAATGGGAGC360    TAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAGTATA420    TTGATTAGTTTTGGAGTACTCGTACGGATTCACTGGCCGTCCTGTTACAACGTCGTGAC479    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 479 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    AAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAAGCCGTGGTTTTTAAA60    AGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAAAA120    GGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGG180    AAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCA240    CGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTACAGACTAAATG300    TCGGTCGGGGAAGATGTATTCTTCTCATAACATATAGTCGGACCTCTCCTTAATGGGAGC360    TAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAGTATA420    TTGATTAGTTTTGGAGTACTCGTACGGATTCACTGGCCGTCCTGTTACAACGTCGTGAC479    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 480 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    AAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAAGCCGTGGTTTTTAAA60    AGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAAAA120    GGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGG180    AAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCA240    CGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTACAGACTAAATG300    TCGGTCGGGACCGTTGATATATGGTTCATAACATATAGTCGGACCTCTCCTTAATGGGAG360    CTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCGAAAGTAT420    ATTGATTAGTTTTGGAGTACTCGTACGGATTCACTGGCCGTCCTGTTACAACGTCGTGAC480    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 487 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    AAGCTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATAAAGCCGTGGTTTTTAAA60    AGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAAAA120    GGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGGGG180    AAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAACCA240    CGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTACAGACTAAATG300    TCGGTCGGGACCGTTGATATATCCCAAACGGTTCATAACATATAGTCGGACCTCTCCTTA360    ATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATGCG420    AAAGTATATTGATTAGTTTTGGAGTACTCGTACGGATTCACTGGCCGTCCTGTTACAACG480    TCGTGAC487    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1044 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    GTCGACCTTTTTAAGTCGGCAAATATCGCATGTTTGTTCGATAGACATCGAGTGGCTTCA60    AAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAA120    AAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGG180    GGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAAC240    CACGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTTCACAGACTA300    AATGTCGGTCGGGGAACAACATGCGATATTGTTCTCATAAGATATAGTCGGACCTCTCCT360    TAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGTATG420    CGAAAGTATATTGATTAGTTTTGGAGTACTCGTCTCGATGATGTTGTTGATTCTTCTAAA480    TCTTTTGTGATTGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCC540    ATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGG600    AAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAA660    AACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAG720    GTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTC780    ACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGATGGT840    GCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATT900    AATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGT960    GGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGT1020    GTCAGGCGATCTTTGTGACTCGAG1044    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1047 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: both    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    GTCGACCTTTTTAAGTCGGCAAATATCGCATGTTTGTTCGATAGACATCGAGTGGCTTCA60    AAAGTTATCAGGCATGCACCTGGTAGCTAGTCTTTAAACCAATAGATTGCATCGGTTTAA120    AAGGCAAGACCGTCAAATTGCGGGAAAGGGGTCAACAGCCGTTCAGTACCAAGTCTCAGG180    GGAAACTTTGAGATGGCCTTGCAAAGGGTATGGTAATAAGCTGACGGACATGGTCCTAAC240    CACGCAGCCAAGTCCTAAGTCAACAGATCTTCTGTTGATATGGATGCAGTTCACAGACTA300    AATGTCGGTCGGGCAAACATGCGATATTTGCCGTTTGTCATAAGATATAGTCGGACCTCT360    CCTTAATGGGAGCTAGCGGATGAAGTGATGCAACACTGGAGCCGCTGGGAACTAATTTGT420    ATGCGAAAGTATATTGATTAGTTTTGGAGTACTCGTCTCGATGATGTTGTTGATTCTTCT480    AAATCTTTTGTGATTGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGAT540    TCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGAT600    TGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAAT660    GAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACG720    AAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGT780    CTCACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGGTGAT840    GGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATAT900    ATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACC960    CGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAAT1020    CGTGTCAGGCGATCTTTGTGACTCGAG1047    __________________________________________________________________________

What is claimed is:
 1. A method for in vivo trans-splicing, said methodcomprising the steps of:(1) providing to a cultured host cell apolynucteotide molecule encoding a trans-splicing Group I ribozyme, thesequence of said ribozyme being a fusion RNA, the sequence of saidfuision RNA comprising:(a) a first RNA sequence, which hybridizes to atarget RNA that encodes a transcription activator protein, and (b) asecond RNA sequence, which is to be trans-spliced into said targetRNA;wherein said polynucleotide molecule is operably linked to atranscription regulatory element which is specifically recognized bysaid transcription activator protein, such that association of saidtranscription activator protein with said transcription regulatoryelement results in activation of transcription of said polyrxucleotidemolecule and production of said trans-splicing ribozyme; (2) providingsaid target RNA in said host cell; and (3) allowing said polynucleotidemolecule to be transcribed and said trans-splicing ribozyme to catalyzetrans-splicing of said second RNA sequence into said target RNA.
 2. Themethod of claim 1, wherein an RNA sequence that hybridizes to said firstRNA sequence is inserted into the P8 helix of a pro-ribozyme.
 3. Amethod for providing a desired genetic sequence to a single host cell invivo, said method comprising:(1) providing to said cultured host cell apolynucleotide molecule encoding a trains-splicing Group I ribozyme, thesequence of said ribozyme being a fusion RNA, the sequence of saidfusion RNA comprising:(a) a first RNA sequence, which hybridizes to atarget RNA that encodes a transcription activator protein, and (b) asecond RNA sequence, which is the desired genetic sequence and is to betrans-spliced into said target RNA;wherein said polynucleotide moleculeis operably linked to a transcription regulatory element which isspecifically recognized by said transcription activator protein, suchthat association of said transcription activator protein with saidtranscription regulatory element results in activation of transcriptionof said polynucleotide molecule and production of said trans-splicingribozyme; wherein said ribozyme possesses catalytic activity against atarget RNA in said host cell, and wherein said ribozyme istrans-splicing said desired genetic sequence; (2) providing said targetRNA in said host cell; and (3) providing conditions that allow saidpolynucleotide molecule to be transcribed and said trans-splicingribozyne to catalyze trans-splicing of said second RNA sequence, whichis the desired genetic sequence, into said target RNA.
 4. The method ofclaim 3, wherein an RNA sequence that hybridizes to said first RNAsequence is inserted into the P8 helix of a pro-ribozyme.
 5. The methodof claims 1 or 3, wherein said transcription activator protein is GAL4;and wherein said first RNA sequence is a sequence that hybridizes toGAL4 RNA.
 6. The method of claims 1 or 3, wherein said second RNAsequence comprises a sequence that encodes a peptide toxic to the hostcell.
 7. The method of claims 1 or 3, wherein said peptide is the DTApeptide.
 8. The method of claims 1 or 3, wherein said DTA peptide is amutant peptide sequence.
 9. The method of claims 1 or 3, wherein saidmutant peptide sequence comprises amino acids encoded by SEQ ID. No. 40.10. The method of claims 1 or 3, wherein said mutant peptide sequencecomprises amino acids encoded by SEQ ID. No.
 41. 11. The method of claim5, wherein said second RNA sequence is a sequence that encodes the DTApeptide.
 12. The method of claim 11, wherein said DTA peptide is amutant peptide sequence.
 13. The method of claim 11, wherein said mutantpeptide sequence comprises amino acids encoded by SEQ ID. No.
 40. 14.The method of claim wherein 11, said mutant peptide sequence comprisesamino acids encoded by SEQ ID. No.
 41. 15. The method of any one ofclaims 1, 3, 2, 4, or 5-14, wherein said polynucleotide molecule is DNA.16. The method of any one of claims 5-14 wherein an RNA sequence thathybridizes to said first RNA sequence is inserted into the P8 helix of apro-ribozyme.
 17. The method of claims 1 or 3, wherein said culturedhost cell is a prokaryotic cell.
 18. The method of claims 1 or 3,wherein said cultured host cell is a eukaryotic cell.
 19. The method ofclaims 1 or 3, wherein said cultured host cell is a plant cell.
 20. Themethod of claims 1 or 3, wherein said cultured host cell is an animalcell.
 21. The method of claim 20, wherein said animal cell isDrosophila.
 22. The method of claim 20, wherein said animal cell is amammalian cell.
 23. The method of claim 22, herein said mammalian cellis a human cell.